An enzyme electrode usually refers to an electrode in which an enzyme is fixed on the surface of an electrode such as a gold electrode, platinum electrode or carbon electrode. Taking advantage of the reaction specificity of the enzyme, the enzyme electrode has been widely used as a biosensor to specifically detect various physiologically active substances. In particular, the enzyme electrode can be used as a glucose sensor for measuring the concentration of glucose in blood as an important marker in diabetes.
Examples of oxidoreductases used for an enzyme electrode include dehydrogenases represented by glucose dehydrogenase (GDH) and oxidases represented by glucose oxidase (GOD). GOD has a high substrate specificity to glucose and is excellent in heat stability. Since mass production of this enzyme is possible, its production cost is lower than other enzymes, which is advantageous. Also, a system using GDH is unlikely to be influenced by oxygen dissolved in a measurement sample. Therefore, even when the measurement is carried under conditions of low oxygen partial pressure or even when the measurement is carried out for a high concentration of sample requiring a large amount of the enzyme, glucose can be precisely measured.
In cases where these oxidoreductases are applied to the enzyme electrode, there has been a problem that a response current value of the electrode is low. Therefore, the inventors of the present invention proposed, in order to improve the response current value of the electrode, an enzyme electrode having an electron transfer protein together with an electron mediator (see Patent Document 2 below).
The electron mediator refers a redox substance such as a non-protein metal complex or an organic compound, the substance being capable of mediating electron transfer from an oxidoreductase to an electrode. Examples thereof include potassium ferricyanide, phenazine methosulfate, ferrocene and derivatives thereof.
The electron transfer protein refers to a protein capable of being reduced by receiving electrons from an electron donor and then oxidized by donating the electrons to an electron acceptor in an oxidation-reduction system in the body. Examples of the electron transfer protein include cytochrome b and cytochrome C, and preferably cytochrome b562 or the like.
In Patent Document 1, an electron transfer protein, together with oxidoreductase, is immobilized on an electrode and thus electron transfer from a the oxidoreductase to the electrode or to an electron mediator can be promoted, thereby obtaining an enzyme electrode with a high response current value.
For the measurement of the concentration of glucose using these enzyme electrodes, in general, a buffer is put into a thermostat cell, and a coenzyme, CaCl2 and the electron mediator are added thereto. The mixture is then kept at a constant temperature. Thereafter, as a working electrode, for example, an enzyme electrode in which an enzyme is immobilized on a carbon electrode is used. And a counter electrode (for example, platinum electrode) and a reference electrode (for example, Ag/AgCl electrode) are used. A constant voltage is applied to the above-mentioned carbon electrode and after an electric current reaches a steady state, a sample containing glucose is added and then an increase in the electric current is measured.
Thus, these conventional methods require the electron mediator to be included in the electrode, to be immobilized on the surface of the electrode, or to be added into the thermostat cell as an aqueous solution. And, the electron mediator needs to be provided separately from the oxidoreductase. Therefore, the process was complicated and there were problems in the cost of mass production.                Patent Document 1: JP 2003-121407 A        Patent Document 2: WO 02/73181        Patent Document 3: WO 2005/023111        